Accuracy of DNA amplification from archival hematological slides for use in genetic biomarker studies.

Author(s): Boyle EB, Steinbuch M, Tekautz T, Gutman JR, Robison LL, Perentesis JP

Publication: Cancer Epidemiol Biomarkers Prev, 1998, Vol. 7, Page 1127-31

PubMed ID: 9865432 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of DNA extraction method on PCR amplification and DNA sequencing results from methanol-fixed stained and unstained bone marrow aspirates from patients with leukemia. The effect of methanol-fixation and storage at room temperature rather than freezing was also investigated in 2 matched specimens. Bone marrow aspirates were collected between 1967 and 1995 and stored at room temperature until use for this study.

   Summary of Findings:

   Proteinase k digestion did not increase DNA extraction success; however, PCR results were inconsistent when DNA was not purified by phenol-chloroform extraction or MicroTurboGen. When DNA was isolated by lysis followed by phenol-chloroform extraction, amplification of one n-ras and/or k-ras locus was possible in 16 of 17 specimens. The authors report that extraction of DNA from unstained, Wright-Giemsa-stained, or Toluidine blue-stained specimens had no effect on PCR success, but note that only one Toluidine blue stained specimen was used. 10 mutations identified in the specimens by sequencing were not reproduced resulting in an artifactual mutation rate of 1 in 3384 bases. In the two cases with matched frozen specimens, sequencing of the frozen specimen confirmed the data from the methanol-fixed specimen.

   Biospecimens

   • Tissue - Bone Marrow

   Preservative Types

   • Other Preservative
   • Frozen

   Diagnoses:

   • Neoplastic - Leukemia
   • Down Syndrome

   Platform:

   Analyte	Technology Platform
   DNA	PCR
   DNA	DNA sequencing

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Frozen Methanol
   Analyte Extraction and Purification	Protein digestion	None Proteinase K
   Analyte Extraction and Purification	Analyte isolation method	Lysis in TEN with SDS Phenol-chloroform None MicroTurboGen
   PCR Specific	Targeted nucleic acid	k-ras N-ras
   PCR Specific	Type of tissue stain	Unstained Wright-Giemsa Toluidine blue

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